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Supplementary Figures 1-2 from Transcription Factor NF-Y Induces Apoptosis in Cells Expressing Wild-Type p53 through E2F1 Upregulation and p53 Activation

Supplementary Methods from Transcription Factor NF-Y Induces Apoptosis in Cells Expressing Wild-Type p53 through E2F1 Upregulation and p53 Activation

Supplementary Figures 5-6 from Transcription Factor NF-Y Induces Apoptosis in Cells Expressing Wild-Type p53 through E2F1 Upregulation and p53 Activation

Millions of people worldwide suffer from visual disabilities as a result of retinal degeneration. Due to the poor regenerative capability of the central nervous system, retinal cell loss is essentially irreversible. Currently available therapies can only decelerate the degenerative process at late stages and are largely ineffective. However, the possibility of using stem cell-based therapy as cell rescue or cell replacement therapy is broadly being explored. While cell rescue is based on the secretion of biologically active molecules by the transplanted cells, cell replacement refers to the possibility of injected stem cells replacing the defective ones either via direct differentiation, transdifferentiation of the transplanted cells, or via cell fusion-mediated reprogramming of retinal cells.

SummaryCell-fusion-mediated somatic-cell reprogramming can be induced in culture; however, whether this process occurs in mammalian tissues remains enigmatic. Here, we show that upon activation of Wnt/β-catenin signaling, mouse retinal neurons can be transiently reprogrammed in vivo back to a precursor stage. This occurs after their spontaneous fusion with transplanted hematopoietic stem and progenitor cells (HSPCs). Moreover, we demonstrate that retinal damage is essential for cell-hybrid formation in vivo. Newly formed hybrids can proliferate, commit to differentiation toward a neuroectodermal lineage, and finally develop into terminally differentiated neurons. This results in partial regeneration of the damaged retinal tissue, with functional rescue. Following retinal damage and induction of Wnt/β-catenin signaling, cell-fusion-mediated reprogramming also occurs after endogenous recruitment of bone-marrow-derived cells in the eyes. Our data demonstrate that in vivo reprogramming of terminally differentiated retinal neurons after their fusion with HSPCs is a potential mechanism for tissue regeneration.

Vision impairments and blindness caused by retinitis pigmentosa result from severe neurodegeneration that leads to a loss of photoreceptors, the specialized light-sensitive neurons that enable vision. Although the mammalian nervous system is unable to replace neurons lost due to degeneration, therapeutic approaches to reprogram resident glial cells to replace retinal neurons have been proposed. Here, we demonstrate that retinal Müller glia can be reprogrammed in vivo into retinal precursors that then differentiate into photoreceptors. We transplanted hematopoietic stem and progenitor cells (HSPCs) into retinas affected by photoreceptor degeneration and observed spontaneous cell fusion events between Müller glia and the transplanted cells. Activation of Wnt signaling in the transplanted HSPCs enhanced survival and proliferation of Müller-HSPC hybrids as well as their reprogramming into intermediate photoreceptor precursors. This suggests that Wnt signaling drives the reprogrammed cells toward a photoreceptor progenitor fate. Finally, Müller-HSPC hybrids differentiated into photoreceptors. Transplantation of HSPCs with activated Wnt functionally rescued the retinal degeneration phenotype in rd10 mice, a model for inherited retinitis pigmentosa. Together, these results suggest that photoreceptors can be generated by reprogramming Müller glia and that this approach may have potential as a strategy for reversing retinal degeneration. This work was supported by an ERC grant (242630-RERE to M.P. Cosma), the Ministerio de Economia y Competitividad y FEDER (SAF2011-28580, and BFU2015-71984, BFU2015-71984-ERC to M.P. Cosma), an AGAUR grant from Secretaria d´Universitats i Investigació del Departament d´Economia i Coneixement de la Generalitat de Catalunya (2014SGR1137 to M.P. Cosma), 976_Velux Stiftung (to M.P. Cosma), TV3’12_120530_ FUNDACIO MARATO TV3, Ferrer International (to M.P. Cosma and D. Sanges), and Subprograma Juan de la Cierva - Ministerio Ciencia e Innovación (to D. Sanges).

The CCAAT-binding transcription factor NF-Y plays a central role in regulating cellular proliferation by controlling the expression of genes required for cell-cycle progression such as cyclin A, cyclin B1, cyclin B2, cdc25A, cdc25C, and cdk1. Here we show that unrestricted NF-Y activity leads to apoptosis in an E2F1- and wild-type p53 (wtp53)-dependent manner. Unrestricted NF-Y activity induced an increase in E2F1 mRNA and protein levels. Furthermore, NF-Y directly bound the E2F1 promoter and this correlated with the appearance of open chromatin marks. The ability of NF-Y to induce apoptosis was impaired in cells lacking E2F1 and wtp53. Moreover, NF-Y overexpression elicited phosphorylation of wt p53Ser18 in an E2F1-dependent manner. Our findings establish that NF-Y acts upstream of E2F1 in p53-mediated apoptosis. Cancer Res; 70(23); 9711–20. ©2010 AACR.

Background/aims The life cycle of hepatitis C virus (HCV) is intimately linked to the lipid metabolism of the host. In particular, HCV exploits the metabolic machinery of the lipoproteins in several steps of its life cycle such as circulation in the bloodstream, cell attachment and entry, assembly and release of viral particles. However, the details of how HCV interacts with and influences the metabolism of the host lipoproteins are not well understood. A study was undertaken to investigate whether HCV directly affects the protein composition of host circulating lipoproteins. Methods A proteomic analysis of circulating very low-, low- and high-density lipoproteins (VLDL, LDL and HDL), isolated from either in-treatment naive HCV-infected patients or healthy donors (HD), was performed using two-dimensional gel electrophoresis and tandem mass spectrometry (MALDI-TOF/TOF). The results obtained were further investigated using in vitro models of HCV infection and replication. Results A decreased level of apolipoprotein A-I (apoA-I) was found in the LDL fractions of HCV-infected patients. This result was confirmed by western blot and ELISA analysis. HCV cellular models (JFH1 HCV cell culture system (HCVcc) and HCV subgenomic replicons) showed that the decreased apoA-I/LDL association originates from hepatic biogenesis rather than lipoprotein catabolism occurring in the circulation, and is not due to a downregulation of the apoA-I protein concentration. The sole non-structural viral proteins were sufficient to impair the apoA-I/LDL association. Functional evidence was obtained for involvement of apoA-I in the viral life cycle such as RNA replication and virion production. The specific siRNA-mediated downregulation of apoA-I led to a reduction in both HCV RNA and viral particle levels in culture. Conclusions This study shows that HCV induces lipoprotein structural modification and that its replication and production are linked to the host lipoprotein metabolism, suggesting apoA-I as a new possible target for antiviral therapy.